Abstract 225: Apolipoprotein A-IV Is a ß3 Integrin Ligand and an Endogenous Inhibitor of Platelets: Novel Mechanisms of Prevention and Treatment for Atherothrombosis

Apolipoprotein A-IV (apoA-IV) is a lipid binding protein secreted by the intestine during dietary lipid absorption. Several clinical studies in different ethnic populations have demonstrated that apoA-IV levels inversely correlate with cardiovascular diseases. However, the roles of apoA-IV in platelet aggregation and atherothrombosis are completely unknown. Here we isolated apoA-IV from plasma with beads coated with human platelet β3 integrin. Using a biomembrane force probe that detects single-molecule interactions, we clearly demonstrated that apoA-IV specifically binds to purified αIIbβ3 on beads and native αIIbβ3 on platelets or Chinese hamster ovary cells. ApoA-IV and αIIbβ3 interaction could be blocked by a monoclonal antibody against β 3 integrin, M1. Importantly, recombinant apoA-IV can competitively block fibrinogen-αIIbβ3 interaction, and specifically inhibited human and mouse platelet aggregation. Consistently, platelet aggregation was enhanced in mice lacking apoA-IV following stimulation with various agonists. In ex vivo perfusion chambers, apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV enhanced ex vivo thrombosis under both low and high shear stresses. Furthermore, in vivo intravital microscopy models revealed that FeCl 3 - and laser-induced thrombosis were enhanced in mice lacking apoA-IV, while transfusion of human or mouse recombinant apoA-IV significantly attenuated this process. To further identify the potential binding sites of apoA-IV for platelet αIIbβ3 integrin, we deleted apoA-IV N-terminal 38 amino acids and/or the C-terminal 41 amino acids. We observed that deletion of the N-terminus abrogated platelet-inhibitory function. Interestingly, we found that mutation of either of two highly conserved aspartic acid (D) residues at positions 5 and 13 abolished or reduced the inhibitory function of apoA-IV, suggesting that D5 and/or D13 participate in a direct protein-protein interaction between apoA-IV and αIIbβ3 integrin. Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a novel link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases.